1. Field of the Invention
The subject of the invention is a process for producing adherent cells to microcarriers, according to which adherent cells are introduced into a culture vessel which contains microcarriers in a culture medium, and a plurality of cell passages is carried out in the same vessel, each time using all or part of the cells of the preceding cell passage for carrying out the next cell passage. The invention also relates to the implementation of this process for the production of biological agents, used in particular to prepare vaccines or drugs.
2. Summary of the Related Art
In the 1980s, the development of the technology of cell culture on microcarriers facilitated the large-scale production of adherent cells and consequently the production of biological agents. The production of adherent cells intended for the production of biological agents for pharmaceutical use must nevertheless observe a certain number of regulatory constraints, among which the prohibition of the use of adherent cells beyond a certain number of “cell passages” because of the risk of morphological and/or biological transformation of the cells. This is in particular the case of cells of the Vero line.
U.S. Pat. No. 4,664,912 describes a process that is convenient at an industrial scale for producing an industrial batch of cells from a cell seed originating from a working cell bank. It is based on a succession of cell passages, each in different bioreactors, the working volumes of which increase during the successive cell passages. This makes it possible each time to increase the amount of microcarriers while at the same time maintaining an optimal concentration of microcarriers in the culture medium that is usually comprised between 1 and 5 g/l. The cell biomass thus increases during the successive cell passages until the desired industrial batch of cells is obtained. The transfer of the cells from one bioreactor to another bioreactor is carried out after having detached the adherent cells from their microcarriers by means of treatment with trypsin and then by blocking the action of the enzyme by introducing serum proteins or serum into the medium so as to preserve as much as possible the integrity of the cells. The cell suspension obtained is then transferred (in the presence or absence of the used microcarriers) into a larger bioreactor which contains a greater amount of naked microcarriers. However, this method of industrial production of adherent cells requires the use and the handling of a large amount of material, which has an impact on the production costs for the biological agents.
In order to reduce the production costs for adherent cells intended for the production of biological agents, EP 1060241 proposes a faster production method that no longer requires the production to be reinitiated from a cell seed originating from a working bank each time it is desired to obtain an industrial batch of cells. The method consists in transferring, after each cell passage, most of the cells (80 to 90% of the cell biomass) to one or more other bioreactors so as to continue to amplify the cell biomass and to constitute an industrial production batch of cells, while the remaining 10 to 20% of the cells are maintained so as to keep a stock “of feeder cells” from which further batches of cells can be produced. This method nevertheless has the following drawbacks:                the cell batches produced display a certain heterogeneity insofar as they do not all have the same number of cell passages.        The maintaining of a stock of “feeder” cells in culture at the time of each transfer operation inevitably leads to an “aging” of the cells which is directly linked to the number of cell passages carried out, and can therefore be used only for a limited period of time for the regulatory reasons already mentioned.        
To avoid the use of a proteolytic enzyme such as trypsin that is detrimental to cell integrity, Ohlson et al in Cytotechnology (1994), vol 14, p 67-80 describes a “bead to bead cell transfer” technology in the absence of any proteolytic enzyme treatment. The close contact between microcarriers covered by adherent cells and naked microcarriers favours the transfer of cells to the naked microcarriers where the cells can proliferate. Therefore, to increase cell growth, naked microcarriers are added to the culture medium containing microcarriers covered by adherent cells while stirring intermittently the medium to favour the contact between the microcarriers. Nevertheless the cell population which is obtained is dissynchronised with cells in various stages of the cell cycle. This can be of major concern for the production of biological agents.